In International Patent Application No. PCT/AU88/00164, it is disclosed that a fusion protein having a foreign protein or polypeptide component fused to the enzyme glutathione-S-transferase (GST), (E.C. 2.5.1.18), preferably to the carboxy-terminal of the enzyme, avoids several of the difficulties associated with known fusion proteins, for instance fusions wherein the foreign protein or polypeptide is expressed as a fusion with E. coli .beta.-galactosidase, in that the GST fusion proteins are generally soluble and can be purified from bacterial lysates under non-denaturing conditions, for example by affinity chromatography on a column of immobilised glutathione. The GST enzyme in the fusion protein may be derived from the parasite helminth Schistosoma japonicum, or it may be derived from other species including humans and other mammals.
The GST fusion proteins disclosed in International Patent Application No. PCT/AU88/00164 may be used as such, since the foreign protein or polypeptide component thereof often retains its antigenicity and functional activity. Alternatively, the fusion protein may be cleaved to provide the foreign protein or polypeptide as a synthesis product, and when the production of such a synthetic protein or polypeptide is desired a cleavable link may be provided in the fusion protein between the glutathione-S-transferase component and the foreign protein or polypeptide component. The cleavable link is preferably one which can be cleaved by a site-specific protease such as thrombin, blood coagulation Factor Xa, or the like.
Expression vectors for use in the expression of such GST fusion proteins are now available commercially and are known as "pGEX vectors". A pGEX vector is an expression vector, more particularly a bacterial plasmid for use in the production of a foreign protein or polypeptide, wherein the vector has inserted therein a nucleotide sequence capable of being expressed as the glutathione-S-transferase enzyme followed by at least one restriction endonuclease recognition site for insertion of a nucleotide sequence capable of being expressed as a foreign protein or polypeptide fused with the COOH-terminus of the glutathione-S-transferase enzyme, optionally with a sequence capable of being expressed as a cleavable link between the enzyme and the foreign protein or polypeptide.
Expression of the GST fusion proteins by the pGEX expression vectors is under the control of the tac promoter which enables inducible, high-level production of these fusion proteins. The pGEX expression vectors also contain the lac Iq gene, so that they can be used in any E. coli strain.
Polypeptides expressed in E. coli as fusions with GST have proven useful for the analysis of protein-DNA and protein-protein interactions. Part of the reason for this is that, in contrast to many other expression systems, the purification of GST fusion proteins involves non-denaturing conditions so that the expressed polypeptide is recovered in a relatively native state and retains at least some of its normal properties.
The present invention provides an improved expression vector system based on the pGEX expression vectors described above, which retains the high expression characteristics of this system but yields a protein product that is not a GST fusion. Thus, the system of this invention has an advantage over the existing pGEX technology in that a high yield of a "native" protein can be obtained without the necessity to cleave away the GST enzyme moiety of a GST fusion protein. This is especially useful where it is demonstrated that GST does not function as a purification aid.